*Target (protein/gene name): Protein Tyrosine Phosphatase (lmo800)
*NCBI Gene # or RefSeq#: 985934 *Protein ID (NP or XP #) or Wolbachia#: CAC99878.1 *Organism (including strain): Listeria monocytogenes
Etiologic Risk Group (see link below): Category B Priority Pathogen according to NIH (National Institute of Allergy and Infectious Diseases) *Disease Information (sort of like the Intro to your Mini Research Write up): Listeria monocytogenes is a gram-positive bacterium that causes listeric infections in humans and other animals. This foodborne pathogen has caused outbreaks of listeriosis in North America and Europe. The symptoms of listeriosis include fever and muscle aches followed by diarrhea and other gastrointestinal problems. By the time of diagnosis, the infection becomes invasive meaning it spreads beyond the gastrointestinal tract. Most often than not the infections are caused by the consumption of contaminated foods. Commonly contaminated foods include meats and dairy products; uncooked, unpasteurized, cooked and processed included. Link to TDR Targets page (if present): N/A Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) http://www.ncbi.nlm.nih.gov/gene/?term=985934 Essentiality of this protein: It is an enzyme that dephosphorylates phosphoserine, phosphthreonine, and phosphotyrosine. iIt contain a functional export signal for secretion or membrane insertion. The lack of lmPTP showed ineffective host colonizes, proving that it plays an important role in virulence of the bacteria. Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Complex of proteins?: Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
In tuberculosis http://iai.asm.org/content/79/6/2489.full
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): -- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information -- links to assay reagents (substrates) pages. --- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 1YWF -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB ---- Query Coverage: 84% ---- Max % Identities: 26% ---- % Positives ---- Chain used for homology:
BlastP analysis with a pairwise alignment between Listeria monocytogenes protein tyrosine phosphatase against Mycobacterium tuberculosis PTP, 1YWF. The Identity is established at 26% and positives are at 47%. Query coverage was 84%.
Current Inhibitors: Expression Information (has it been expressed in bacterial cells): Purification Method: Image of protein (PyMol with features delineated and shown separately): Pymol representation of the Crystal Structure of Mycobacterium Tuberculosis Protein Tyrosine Phosphatase PtpB (1YWF:PDB) shown as lines colored by element with carbon as green. Active site shown as sticks colored by element with carbon as Cyan. Ligand PO4 shown in sticks colored by element with carbon as Pink.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MKNWVKVTGAGVLSATLLLGGCGAQSEEKAEANVKTEQTLKPGSQIKLEGAVNVRDLGGYKTTDGLTIKPHKLIRSAELANLSDSDKKKLVNTYDLSHIVDFRTSSEVATKPDPKLTDVDYTHDSVMKDNGTSTSTQDLTASLAKMDNPETFLINANKSFITDETSIQAYKDFFDILLANQDGSVLWHCTAGKDRAGFGTALVLSALGVDKNTVIDDYMLSNKYRADENKKAIEAVAAKTDNKKVIDGMTAVMEVRESYINAAFDEINAKYGSMDNFLKEKLGLTDAKKEQLKKAYLY
*length of your protein in Amino Acids 296 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 32585.8 kiloDaltons Molar Extinction coefficient of your protein at 280 nm wavelength: 26025 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only): ATGAAAAATTGGGTAAAAGTAACAGGAGCAGGGGTACTAAGCGCGACTTTACTATTAGGTGGATGCGGGG CGCAGTCAGAAGAAAAAGCAGAAGCAAATGTTAAAACCGAGCAAACACTCAAACCAGGAAGCCAAATTAA ATTAGAAGGCGCTGTAAATGTCCGGGACTTAGGCGGATACAAAACAACGGATGGACTAACCATTAAGCCA CATAAACTCATTAGAAGTGCCGAACTCGCTAACTTAAGTGATTCGGATAAAAAGAAACTCGTAAATACAT ATGATTTGTCTCATATAGTTGATTTCCGAACAAGTTCAGAAGTCGCAACGAAACCAGATCCGAAACTCAC AGATGTAGACTATACGCACGATTCTGTGATGAAAGATAATGGAACATCTACAAGCACACAAGATTTAACT GCTAGCCTAGCCAAAATGGATAATCCAGAGACATTTCTCATTAATGCTAATAAGAGCTTTATTACAGATG AAACTTCGATACAAGCCTATAAAGATTTTTTTGATATACTACTAGCAAACCAAGATGGTTCTGTTCTTTG GCACTGTACAGCTGGAAAAGACCGAGCTGGATTTGGAACCGCCCTCGTTCTTTCAGCGTTAGGTGTGGAT AAAAACACGGTCATTGACGATTATATGCTGTCCAATAAATATCGTGCTGACGAAAATAAAAAAGCAATTG AAGCCGTTGCAGCAAAAACAGATAATAAAAAAGTGATTGATGGAATGACAGCCGTAATGGAAGTTCGTGA ATCTTATATCAATGCAGCCTTCGATGAAATTAATGCAAAATATGGTTCGATGGACAACTTTTTAAAAGAA AAACTCGGACTAACCGATGCTAAAAAAGAACAACTAAAAAAAGCATATCTTTATTAA *GC% Content for gene: 37.9%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): 1 ATGAAAAACTGGGTTAAGGTTACCGGTGCGGGTGTTCTCTCTGCGACCCTGCTCCTGGGC 61 GGCTGCGGTGCGCAGTCTGAAGAGAAGGCTGAAGCGAACGTCAAAACCGAACAGACCCTG 121 AAACCGGGCTCTCAGATCAAACTGGAAGGTGCGGTAAACGTTCGTGATCTGGGTGGCTAC 181 AAGACCACCGACGGCCTGACCATCAAGCCGCACAAACTGATCCGTTCTGCGGAACTCGCG 241 AATCTGAGCGACTCTGACAAGAAGAAACTGGTTAACACCTACGACCTGTCTCACATCGTT 301 GACTTCCGTACCTCTTCTGAAGTTGCGACCAAACCGGACCCGAAACTGACCGACGTAGAC 361 TATACCCACGATAGCGTTATGAAGGACAACGGTACCAGCACGTCTACCCAGGATCTGACC 421 GCCTCTCTGGCTAAAATGGATAACCCGGAAACCTTCCTGATCAACGCAAACAAGTCTTTC 481 ATTACTGACGAAACGTCTATCCAGGCGTACAAGGACTTCTTCGACATCCTGCTGGCGAAC 541 CAGGACGGTTCTGTTCTGTGGCACTGCACGGCGGGTAAAGATCGTGCGGGCTTCGGCACC 601 GCGCTGGTCCTGTCTGCTCTCGGTGTAGACAAAAACACCGTTATTGACGACTACATGCTG 661 TCTAACAAATACCGTGCGGACGAAAACAAGAAAGCTATCGAAGCGGTTGCGGCGAAAACG 721 GACAACAAAAAAGTTATCGACGGTATGACGGCGGTAATGGAAGTACGTGAATCTTACATT 781 AACGCGGCGTTCGACGAGATTAATGCGAAATACGGCTCTATGGACAACTTTCTGAAAGAA 841 AAGCTGGGTCTGACGGACGCGAAGAAAGAACAACTGAAGAAGGCGTATCTGTACTAA *GC% Content for gene (codon optimized): 51.5%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*Target (protein/gene name): Protein Tyrosine Phosphatase (lmo800)
*NCBI Gene # or RefSeq#: 985934*Protein ID (NP or XP #) or Wolbachia#: CAC99878.1
*Organism (including strain): Listeria monocytogenes
Etiologic Risk Group (see link below): Category B Priority Pathogen according to NIH (National Institute of Allergy and Infectious Diseases)*Disease Information (sort of like the Intro to your Mini Research Write up):
Listeria monocytogenes is a gram-positive bacterium that causes listeric infections in humans and other animals. This foodborne pathogen has caused outbreaks of listeriosis in North America and Europe. The symptoms of listeriosis include fever and muscle aches followed by diarrhea and other gastrointestinal problems. By the time of diagnosis, the infection becomes invasive meaning it spreads beyond the gastrointestinal tract. Most often than not the infections are caused by the consumption of contaminated foods. Commonly contaminated foods include meats and dairy products; uncooked, unpasteurized, cooked and processed included.
Link to TDR Targets page (if present): N/A
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
http://www.ncbi.nlm.nih.gov/gene/?term=985934
Essentiality of this protein:
It is an enzyme that dephosphorylates phosphoserine, phosphthreonine, and phosphotyrosine. iIt contain a functional export signal for secretion or membrane insertion. The lack of lmPTP showed ineffective host colonizes, proving that it plays an important role in virulence of the bacteria.
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Complex of proteins?:
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
In tuberculosis http://iai.asm.org/content/79/6/2489.full
*EC#: 3.1.3.48
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.3.48
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1YWF
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 84%
---- Max % Identities: 26%
---- % Positives
---- Chain used for homology:
BlastP analysis with a pairwise alignment between Listeria monocytogenes protein tyrosine phosphatase against Mycobacterium tuberculosis PTP, 1YWF. The Identity is established at 26% and positives are at 47%. Query coverage was 84%.
Current Inhibitors:
Expression Information (has it been expressed in bacterial cells):
Purification Method:
Image of protein (PyMol with features delineated and shown separately):
Pymol representation of the Crystal Structure of Mycobacterium Tuberculosis Protein Tyrosine Phosphatase PtpB (1YWF:PDB) shown as lines colored by element with carbon as green. Active site shown as sticks colored by element with carbon as Cyan. Ligand PO4 shown in sticks colored by element with carbon as Pink.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MKNWVKVTGAGVLSATLLLGGCGAQSEEKAEANVKTEQTLKPGSQIKLEGAVNVRDLGGYKTTDGLTIKPHKLIRSAELANLSDSDKKKLVNTYDLSHIVDFRTSSEVATKPDPKLTDVDYTHDSVMKDNGTSTSTQDLTASLAKMDNPETFLINANKSFITDETSIQAYKDFFDILLANQDGSVLWHCTAGKDRAGFGTALVLSALGVDKNTVIDDYMLSNKYRADENKKAIEAVAAKTDNKKVIDGMTAVMEVRESYINAAFDEINAKYGSMDNFLKEKLGLTDAKKEQLKKAYLY
*length of your protein in Amino Acids 296
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
32585.8 kiloDaltons
Molar Extinction coefficient of your protein at 280 nm wavelength: 26025
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
ATGAAAAATTGGGTAAAAGTAACAGGAGCAGGGGTACTAAGCGCGACTTTACTATTAGGTGGATGCGGGG CGCAGTCAGAAGAAAAAGCAGAAGCAAATGTTAAAACCGAGCAAACACTCAAACCAGGAAGCCAAATTAA ATTAGAAGGCGCTGTAAATGTCCGGGACTTAGGCGGATACAAAACAACGGATGGACTAACCATTAAGCCA CATAAACTCATTAGAAGTGCCGAACTCGCTAACTTAAGTGATTCGGATAAAAAGAAACTCGTAAATACAT ATGATTTGTCTCATATAGTTGATTTCCGAACAAGTTCAGAAGTCGCAACGAAACCAGATCCGAAACTCAC AGATGTAGACTATACGCACGATTCTGTGATGAAAGATAATGGAACATCTACAAGCACACAAGATTTAACT GCTAGCCTAGCCAAAATGGATAATCCAGAGACATTTCTCATTAATGCTAATAAGAGCTTTATTACAGATG AAACTTCGATACAAGCCTATAAAGATTTTTTTGATATACTACTAGCAAACCAAGATGGTTCTGTTCTTTG GCACTGTACAGCTGGAAAAGACCGAGCTGGATTTGGAACCGCCCTCGTTCTTTCAGCGTTAGGTGTGGAT AAAAACACGGTCATTGACGATTATATGCTGTCCAATAAATATCGTGCTGACGAAAATAAAAAAGCAATTG AAGCCGTTGCAGCAAAAACAGATAATAAAAAAGTGATTGATGGAATGACAGCCGTAATGGAAGTTCGTGA ATCTTATATCAATGCAGCCTTCGATGAAATTAATGCAAAATATGGTTCGATGGACAACTTTTTAAAAGAA
AAACTCGGACTAACCGATGCTAAAAAAGAACAACTAAAAAAAGCATATCTTTATTAA
*GC% Content for gene:
37.9%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
1 ATGAAAAACTGGGTTAAGGTTACCGGTGCGGGTGTTCTCTCTGCGACCCTGCTCCTGGGC
61 GGCTGCGGTGCGCAGTCTGAAGAGAAGGCTGAAGCGAACGTCAAAACCGAACAGACCCTG
121 AAACCGGGCTCTCAGATCAAACTGGAAGGTGCGGTAAACGTTCGTGATCTGGGTGGCTAC
181 AAGACCACCGACGGCCTGACCATCAAGCCGCACAAACTGATCCGTTCTGCGGAACTCGCG
241 AATCTGAGCGACTCTGACAAGAAGAAACTGGTTAACACCTACGACCTGTCTCACATCGTT
301 GACTTCCGTACCTCTTCTGAAGTTGCGACCAAACCGGACCCGAAACTGACCGACGTAGAC
361 TATACCCACGATAGCGTTATGAAGGACAACGGTACCAGCACGTCTACCCAGGATCTGACC
421 GCCTCTCTGGCTAAAATGGATAACCCGGAAACCTTCCTGATCAACGCAAACAAGTCTTTC
481 ATTACTGACGAAACGTCTATCCAGGCGTACAAGGACTTCTTCGACATCCTGCTGGCGAAC
541 CAGGACGGTTCTGTTCTGTGGCACTGCACGGCGGGTAAAGATCGTGCGGGCTTCGGCACC
601 GCGCTGGTCCTGTCTGCTCTCGGTGTAGACAAAAACACCGTTATTGACGACTACATGCTG
661 TCTAACAAATACCGTGCGGACGAAAACAAGAAAGCTATCGAAGCGGTTGCGGCGAAAACG
721 GACAACAAAAAAGTTATCGACGGTATGACGGCGGTAATGGAAGTACGTGAATCTTACATT
781 AACGCGGCGTTCGACGAGATTAATGCGAAATACGGCTCTATGGACAACTTTCTGAAAGAA
841 AAGCTGGGTCTGACGGACGCGAAGAAAGAACAACTGAAGAAGGCGTATCTGTACTAA
*GC% Content for gene (codon optimized):
51.5%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Forward Primer: TACTTCCAATCCATGAAAAACTGGGTTAAAGTTAC
Reverse Primer: TATCCACCTTTACTGTTAGTACAGGTAAGCTTTCT